The present invention relates to a novel ligand polypeptide for the G protein-coupled receptor protein and a DNA comprising a DNA encoding the ligand polypeptide.
Many hormones and neurotransmitters mediate Of biological functions through specific receptors present on the cell membrane. Many of these receptors engage themselves in the intracellular transduction of signals through activation of the coupled guanine nucleotide-binding protein (hereinafter sometimes referred to briefly as G protein) and have the common structure comprising 7 transmembrane domains. Therefore, these receptors are collectively referred to as G protein-coupled receptor or 7-transmembrane receptor.
One of the pathways to modulate biological functions mediated by such hormones or neurotransmitters through G protein-coupled receptors is the hypothalamo-pituitary system. Thus, the secretion of pituitary hormone from the hypophysis is controlled by hypothalamic hormones (pituitatropic releasing factor) and the functions of the target cells or organs are regulated through the pituitary hormones released into the circulation. This pathway carries out functional modulations of importance to the living body, such as homeostasis and regulation of the reproduction, development, metabolism and growth of individuals. The secretion of pituitary hormones is controlled by a positive feedback or a negative feedback mechanism involving hypothalamic hormone and the peripheral hormone secreted from the target endocrine gland. The various receptor proteins present in the hypophysis are playing a central role in the regulation of the hypothalamus-pituitary system.
Meanwhile, it is known that these hormones and factors as well as their receptors are not localized in the hypothalamus-pituitary system but are broadly distributed in the brain. Therefore, it is suspected that, in the central nervous system, this substance called hypothalamus hormone is functioning as a neurotransmitter or a neuromodulator. Moreover, the substance is distributed in peripheral tissues as well and thought to be playing important roles in the respective tissue.
The pancreas is playing a crucial role in the carbohydrate metabolism by secreting glucagon and insulin as well as digestive juice. While insulin is secreted from the pancreatic xcex2 cells, its secretion is mainly stimulated by glucose. However, it is known that xcex2 cells have a variety of receptors and the secretion of insulin is controlled by a number of factors in addition to glucose as well as peptide hormones, e.g. galanine, somatostatin, gastric inhibitory polypeptide, glucagon, amyrin, etc.; sugars, e.g. mannose etc.; amino acids, and neurotransmitters, among others.
The means only heretofore available for identifying ligands for said G protein-coupled receptor proteins is estimation from the homology in primary structure of G protein-coupled receptor proteins.
Recently, investigation for novel opioid peptides by introducing a cDNA coding for a receptor protein which a ligand is unknown, i.e. an orphan G protein-coupled receptor protein, into animal cells have been reported (Reinsheid, R. K. et al., Science, 270, 792-35 794, 1995, Menular, J.-C., et al., Nature 377, 532-535, 1995). However, in view of similarities to known G protein-coupled receptor proteins and tissue distributions, it could be easily anticipated in these cases that the ligand would be belonging to the family of opioid peptides. The history of research and development in the realm of substances acting on the living body through the opioid receptor dates back to many years ago and various antagonists and agonists had been developed. Therefore, among the compounds artificially synthesized, an agonist of the receptor was picked out and, using it as a probe, expression of the receptor in the receptor cDNA-transfected cells was verified. Then, a search was made for an activator of the intracellular signal transduction which was similar to the agonist, the activator so found was purified, and the structure of the ligand was determined. However, when the homology of an orphan receptor to known G protein-coupled receptor proteins is low, it was very difficult to predict its ligand.
Ligands for orphan G protein-coupled receptors expressed in the hypophysis, central nervous system, and pancreatic xcex2 cells are considered to be useful for developing medicines, but their structures and functions have not been elucidated as yet.
Employing a cell in which a cDNA coding for orphan G protein-coupled receptor protein hare been expressed by a suitable means and using measurement of a specific cell stimulation activity exemplified by a signal transduction activity as an indicator, the inventors of the present invention succeeded in screening a polypeptide which said receptor protein recognizes as a ligand.
Furthermore, the inventors found that a compound can be screened which is capable of changing the binding activity of this ligand which is an activating factor to said receptor protein.
The present invention, therefore, relates to
(1) A polypeptide which comprises an amino acid sequence represented by SEQ ID NO:73 or its substantial equivalent thereto, or its amide or ester, or a salt thereof.
(2) The polypeptide as described in (1) above, which comprises the amino acid sequence represented by SEQ ID NO:3, SEC ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, or SEQ ID NO:66.
(3) The polypeptide as described in (1) above, which comprises the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO:59.
(4) A partial peptide of the polypeptide as described in (1) above its amide or ester, or a salt thereof.
(5) A DNA which comprises a DNA having a nucleotide sequence coding for the polypeptide as described in (1) above or the partial peptide as described in (4) above.
(6) The DNA as described in (5) above which comprises a nucleotide sequence represented by SEQ ID NO:2, SEQ ID NO:1l, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:18, SEQ ID NO:46, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72.
(7) A recombinant vector comprising the DNA as described in (5) above.
(8) A transformant carrying the DNA as described in (5) above or the recombinant vector as described (7) above.
(9) A method for producing the polypeptide as described in (1) above or the partial peptide as described in (4) above, which comprises culturing the transformant as described in (8) above.
(10) A pharmaceutical composition containing the polypeptide, its amide or ester as described in (1) above, or a pharmaceutically acceptable salt thereof.
(11) A pharmaceutical composition containing the partial peptide peptide, its amide or ester as described in (4) above, or a pharmaceutically acceptable salt thereof.
(12) A pharmaceutical composition containing the DNA as described in (5) above.
(13) The pharmaceutical composition as described in (10), (11), or (12) above, which is a pituitary function modulator.
(14) The pharmaceutical composition as described in (10), (11), or (12) above, which is a central nervous system function modulator.
(15) The pharmaceutical composition as described in (10), (11), or (12) above, which is a pancreatic function modulator.
(16) An antibody against the polypeptide as described in (1) above or against the partial peptide as described in (4) above.
(17) A screening method for a compound capable of changing the binding activity of the polypeptide as described in (1) above or the partial peptide as described in (4) above, with a receptor protein comprising an amino acid sequence represented by SEQ ID NO:21 or its partial, peptide or its substantial equivalent thereto, or a salt thereof, which comprises making a comparison between: (i) at lease one case where said polypeptide as described in (1) above or the partial peptide as described in (4) above is contacted with a receptor protein comprising an amino acid sequence represented by SEQ ID:21 or its partial peptide or its substantial equivalent thereto, or a salt thereof, and (ii) at least one case where said polypeptide as described in (1) above or the partial peptide as described in (4) above together with a sample to be tested in contacted with protein comprising an amino acid sequence represented by SEQ ID NO:21 or its partial peptide or its substantial equivalent thereto, or a salt thereof.
(18) A kit for screening for a compound capable of changing the binding activity of the polypeptide as described in (1) above or the partial peptide as described in (4) above with a receptor protein comprising an amino acid sequence represented by SEQ ID NO:21 or its partial peptide or its substantial equivalent thereto, or a salt thereof.
(19) A compound capable of changing the binding activity of the polypeptide as described in (1) or the partial peptide as described in (4) with a receptor protein comprising an amino acid sequence represented by SEQ ID NO:21 or its partial peptide or its substantial equivalent thereto, or a salt thereof.
(20) A G protein-coupled receptor protein which recognizes the polypeptide as described in (1) above or the partial peptide as described in (4) above as a ligand, or a salt thereof.
The present invention further provides:
(21) the polypeptide as described in (1) above, or its amide or ester, or a salt thereof, which comprises an amino acid sequence selected from the group consisting of an amino acid sequence of SEQ ID NO:73, amino acid sequences wherein 1 to 15 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues are deleted from the amino acid sequence of SEQ ID NO:73, amino acid sequences wherein 1 to 80 amino acid residues, preferably 1 to 50 amino acid residues, more preferably 1 to 10 amino acid residues are added to the amino acid sequence of SEQ ID NO:73, and amino acid sequences wherein 1 to 15 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid resides in the amino acid sequence of SEQ ID NO:73 are substituted with one or more other amino acid residues;
(22) the polypeptide as described in (1) above, which comprises an amino acid sequence wherein the peptide of SEQ ID NO:74 is added to the N-terminus of the polypeptide comprising the amino acid sequence of SEQ ID NO:73;
(23) the polypeptide as described in (1) above, which in derived from bovine, rat or human; and
(24) the pharmaceutical composition described in (10), (11) or (12) above, which is a therapeutic and/or prophylactic agent for dementia, depression (melancholia), hyperkinetic (microencephalo-pathy) syndrome, disturbance of consciousness, anxiety syndrome, schizophrenia, horror, growth hormone secretory disease, hyperphagia, polyphagia, hypercholesterolemia, hyperglyceridemia, hyperlipemia, hyporprolactinemia, diabetes, cancer, pancreatitis, renal disease, Turner""s syndrome, neurosis, rheumatoid arthritis, spinal injury, transient brain ischemia, amyotrophic lateral sclerosis, acute myocardial infarction, spinocerebellar degeneration, bone fracture, trauma, atopic dermatitis, osteoporosis, asthma, epilepsy, infertility and/or oligogalactia.
Referring to the G protein-coupled receptor protein for the ligand polypeptide in accordance with the present invention, the invention specifically provides:
(25) the G protein-coupled receptor protein described in (20) or a salt thereof, which comprises an amino acid sequence represented by SEQ ID NO:19 or its substantial equivalent thereto or/and an amino acid sequence represented by SEQ ID NO:20 or its substantial equivalene thereto;
(26) the G protein-coupled receptor protein described in (25) above or a salt thereof, which comprises an amino acid sequence represented by SEQ ID NO:21 or its substantial equivalent thereto;
(27) the G protein-coupled receptor protein described in (25) above or a salt thereof, which comprises an amino acid sequence represented by SEQ ID NO:22 or its substantial equivalent thereto;
(28) the G protein-coupled receptor protein described in (25) above or a salt thereof, which comprises an amino acid sequence represented by SEQ ID NO:23 or its substantial equivalent thereto;
(29) a partial peptide of any of the G protein-coupled receptor proteins described in (25)-(28) above or a salt thereof;
(30) a DNA which comprises a DNA having a nucleotide sequence coding for the G protein-coupled receptor protein described in (25) above;
(31) a DNA which comprises a DNA having a nucleotide sequence coding for the G protein-coupled receptor protein described in (26) above;
(32) a DNA which comprises a DNA having a nucleotide sequence coding for the G protein-coupled receptor protein described in (27) above;
(33) a DNA which comprises a DNA having a nucleotide sequence coding for the G protein-coupled receptor protein described in (28) above;
(34) the DNA described in (30) above, which comprises the nucleotide sequence of SEQ ID NO:24 or the nucleotide sequence of SEQ ID NO:25;
(35) the DNA described in (31) above, which comprises the nucleotide sequence of SEQ ID NO:26;
(36) the DNA described in (32) above, which comprises the nucleotide sequence of SEQ ID NO:27;
(37) the DNA described in (33) above, which comprises the nucleotide sequence of SEQ ID NO:28;
(38) a recombinant vector comprising any of the DNAs described in (30)-(33) above;
(39) a transformant carrying the recombinant vector described in (38) above;
(40) a method for producing the G protein-coupled receptor protein described in (28) above or a salt thereof, which comprises culturing the transformant of (39) to produce said G protein-coupled receptor protein on the cell membrane of the transformant;
(41) an antibody to any of the G protein-coupled receptor protein described in (25)-(28) above or a salt thereof, or the partial peptide described in (29) above or a salt thereof.
To be further specific, the G protein-coupled receptor protein relates to:
(42) the G protein-coupled receptor protein described in (25) above or a salt thereof, wherein the protein comprises (i) an amino acid sequence selected from the group consisting of an amino acid sequence of SEQ ID NO:19, amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues are deleted from the amino acid sequence of SEQ ID NO:19, amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues are added to the amino acid sequence of SEQ ID NO:19, and amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues in the amino acid sequence of SEQ ID NO:19 are substituted with one or more than amino acid residues and/or (ii) an amino acid sequence selected from the group consisting of an amino acid sequence of SEQ ID NO:20, amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues are deleted from the amino acid sequence of SEQ ID NO:20, amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues are, added to the amino acid sequence of SEQ ID NO:20, and amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues in the amino acid sequence of SEQ ID NO:20 are substituted with one or more other amino acid residues;
(43) the G protein-coupled receptor protein described in (26) above or a salt thereof, wherein the protein comprises an amino acid sequence selected from the group consisting of an amino acid sequence of SEQ ID NO:21, amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues are deleted from the amino acid sequence of SEQ ID NO:21, amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues are added to the amino acid sequence of SEQ ID NO:21, and amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues in the amino acid sequence of SEQ ID NO:21 are substituted with one or more other amino acid residues;
(44) the G protein-coupled receptor protein described in (27) above or a salt thereof wherein the protein comprises an amino acid sequence selected from the group consisting of an amino acid sequence of SEQ ID NO:22, amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues are deleted from the amino acid sequence of SEQ ID NO:22, amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues are added to the amino acid sequence of SEQ ID NO:22, and amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues in the amino acid sequence of SEQ ID NO:22 are substituted with one or more other amino acid residues;
(45) the G protein-coupled receptor protein described in (28) above or a salt thereof, wherein the protein comprises an amino acid sequence selected from the group consisting of an amino acid sequence of SEQ ID NO:23, amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues are deleted from the amino acid sequence of SEQ ID NO:23, amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues are added to the amino acid sequence of SEQ ID NO:23, and amino acid sequences wherein 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues in the amino acid sequence of SEQ ID NO:23 are substituted with one or more other amino acid residues.
As used herein the term xe2x80x9csubstantial equivalent(s)xe2x80x9d means that the activity of the protein, e.g., nature of the binding activity of the ligand and the receptor and physical characteristics are substantially the same. Substitutions, deletions or insertions of amino acids often do not produce radical changes in the physical and chemical characteristics of a polypeptide, in which case polypeptides containing the substitution, deletion, or insertion would be considered to be substantially equivalent to polypeptides lacking the substitution, deletion, or insertion. Substantially equivalent substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. The non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.